Nerve injury dramatically increases NPY expression in sensory neurons and NPY receptor-mediated analgesia, yet this is underappreciated as a potential pain relief mechanism that might prevent the transition from acute to chronic pain. Among 35 original research articles, reviews, and a book published during the previous funding cycle (28 as first or senior authors), our work published in PNAS discovered that intrathecal NPY receptor antagonists or conditional genetic NPY depletion reinstated hyperalgesia, even when applied months after nerve injury or inflammation. Our overall hypothesis is that injury induces a sustained release of NPY and activation of NPY Y1 receptor signaling that opposes the transition to a chronic state of lasting pain vulnerability. However, whether tissue- or nerve-injury induced increases in spinal NPY Y1 receptor (Y1R) signaling is associated with a tonic increase in the releasable pool of NPY has been difficult to determine. To address this gap, we developed a new in situ assay of functional NPY release (Y1R internalization). Our new data indicate that Y1R internalization is greater with nerve injury after high frequency electrical stimulation of dorsal horn slices or after in vivo non-noxious stimulation of the hindpaw. Specific Aim 1 will use this novel method to test the hypothesis that non-noxious stimulus-evoked release of NPY from A-fibers will remain primed for weeks and even months after nerve injury. We now report that NPY produces an outward current and inhibits action potentials evoked by dorsal root stimulation (DRS) in Y1-EGFP labeled neurons. To test the hypothesis that injury increases synaptic inhibition by NPY, Specific Aim 2A will patch-clamp lamina II neurons and conduct Fura-2 [Ca2+]i imaging at peak of hyperalgesia after injury. We predict that Y1R agonists will inhibit stimulus-evoked responses and their concentration-response curves will be left-shifted by injury. Our progress with NPY saporin-conjugated neurotoxin indicates that Y1R-expressing dorsal horn neurons contribute to neuropathic pain, and new double-label immunohistochemistry and patch-clamp electrophysiology data in Y1R-EGFP mice support the concept that Y1R-positive neurons are excitatory. To test the hypothesis that Y1R-expressing neurons maintain LS after inflammation or nerve injury, but are held in check by NPY, Specific Aim 2B will evaluate the effects of Y1 receptor antagonists on [Ca2+]i mobilization and NMDA or AMPA receptor currents in Y1R-expressing neurons upon dorsal root stimulation or laser-directed NMDA/AMPA uncaging. Specific Aim 3 will then use an intrathecal pharmacology approach to determine whether NPY silences pronociceptive GluN? AC1? Epac signaling on Y1R-expressing dorsal horn neurons and TRPA1? TRPV1 signaling on central afferents terminals. Completion of this project will bring us closer to our long-term goal of alleviating chronic pain by either: a) facilitating endogenous NPY analgesia, thus restricting pain within a state of remission; or b) extinguishing spinal neuron sensitization altogether, for example with a selective AC1 or Epac1 inhibitor.